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CpG promoter methylation of the ALKBH3 alkylation repair gene in breast cancer

CpG promoter methylation of the ALKBH3 alkylation repair gene in breast cancer

Titill: CpG promoter methylation of the ALKBH3 alkylation repair gene in breast cancer
Höfundur: Stefansson, Olafur   orcid.org/0000-0002-3384-6817
Hermanowicz, Stefan
van der Horst, Jasper
Hilmarsdóttir, Hólmfríður
Staszczak, Zuzanna
Jónasson, Jón Gunnlaugur
Tryggvadottir, Laufey   orcid.org/0000-0001-8067-9030
Guðjónsson, Þorkell
Sigurðsson, Stefán
Útgáfa: 2017-07-05
Tungumál: Enska
Umfang: 469
Háskóli/Stofnun: Háskóli Íslands
University of Iceland
Svið: Heilbrigðisvísindasvið (HÍ)
School of Health Sciences (UI)
Deild: Læknadeild (HÍ)
Faculty of Medicine (UI)
Birtist í: BMC Cancer;17(1)
ISSN: 1471-2407
DOI: 10.1186/s12885-017-3453-8
Efnisorð: Erfðafræði; Krabbameinsrannsóknir; Brjóstakrabbamein
URI: https://hdl.handle.net/20.500.11815/365

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Stefansson, O. A., Hermanowicz, S., van der Horst, J., Hilmarsdottir, H., Staszczak, Z., Jonasson, J. G., . . . Sigurdsson, S. (2017). CpG promoter methylation of the ALKBH3 alkylation repair gene in breast cancer. BMC Cancer, 17(1), 469. doi:10.1186/s12885-017-3453-8


Background DNA repair of alkylation damage is defective in various cancers. This occurs through somatically acquired inactivation of the MGMT gene in various cancer types, including breast cancers. In addition to MGMT, the two E. coli AlkB homologs ALKBH2 and ALKBH3 have also been linked to direct reversal of alkylation damage. However, it is currently unknown whether ALKBH2 or ALKBH3 are found inactivated in cancer. Methods Methylome datasets (GSE52865, GSE20713, GSE69914), available through Omnibus, were used to determine whether ALKBH2 or ALKBH3 are found inactivated by CpG promoter methylation. TCGA dataset enabled us to then assess the impact of CpG promoter methylation on mRNA expression for both ALKBH2 and ALKBH3. DNA methylation analysis for the ALKBH3 promoter region was carried out by pyrosequencing (PyroMark Q24) in 265 primary breast tumours and 30 proximal normal breast tissue samples along with 8 breast-derived cell lines. ALKBH3 mRNA and protein expression were analysed in cell lines using RT-PCR and Western blotting, respectively. DNA alkylation damage assay was carried out in cell lines based on immunofluorescence and confocal imaging. Data on clinical parameters and survival outcomes in patients were obtained and assessed in relation to ALKBH3 promoter methylation. Results The ALKBH3 gene, but not ALKBH2, undergoes CpG promoter methylation and transcriptional silencing in breast cancer. We developed a quantitative alkylation DNA damage assay based on immunofluorescence and confocal imaging revealing higher levels of alkylation damage in association with epigenetic inactivation of the ALKBH3 gene (P = 0.029). In our cohort of 265 primary breast cancer, we found 72 cases showing aberrantly high CpG promoter methylation over the ALKBH3 promoter (27%; 72 out of 265). We further show that increasingly higher degree of ALKBH3 promoter methylation is associated with reduced breast-cancer specific survival times in patients. In this analysis, ALKBH3 promoter methylation at >20% CpG methylation was found to be statistically significantly associated with reduced survival (HR = 2.3; P = 0.012). By thresholding at the clinically relevant CpG methylation level (>20%), we find the incidence of ALKBH3 promoter methylation to be 5% (13 out of 265). Conclusions ALKBH3 is a novel addition to the catalogue of DNA repair genes found inactivated in breast cancer. Our results underscore a link between defective alkylation repair and breast cancer which, additionally, is found in association with poor disease outcome.


Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated


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